Holding it together
The bacterium Deinococcus radiodurans can tolerate blasts of radiation that are lethal to all other organisms. Its ability to survive under radiation stress has made engineering the bacterium to detoxify pollutants in sites containing mixed metal, organic, and radioactive toxic wastes a promising approach for cleanup of these sites. Now researchers at the Weizmann Institute of Science (Rehovot, Israel), the National Cancer Institute (Bethesda, MD), and the Uniformed Services University of the Health Sciences (Bethseda, MD) suggest that the unusual packaging of the bacterial genome may contribute to its extreme radioresistance (Science 299, 254–256, 2003). After exposure to radiation, an organism's genome often breaks apart into pieces. Surprisingly, D. radiodurans does not contain any special DNA repair enzymes that could mend these breaks. Instead, the new evidence indicates that a tight and ordered packaging of its genome into ring-like structures allows the bug to withstand high doses of radiation. The structures were visible in DNA labeling and microscopy experiments. The researchers suggest that within these structures the free ends of the broken DNA are held together allowing them to be joined correctly without a template. MS
21 and counting
A paper in the Journal of the American Chemical Society (125, in press, 2003) reports the creation of an Escherichia coli strain capable of synthesizing one more amino acid than the normal complement of 20. Researchers at the Scripps Research Institute (La Jolla, CA), led by Peter Schultz, engineered the bacterium with a unique p-aminophenylalanine synthetase and cognate tRNA. The engineered strain could synthesize p-aminophenylalanine from basic carbon sources and incorporate the unnatural amino acid into proteins using an amber nonsense codon. Electrophoretic and mass spectrometric analysis of myoglobin produced by the bacterium demonstrated that p-aminophenylalanine incorporates into the protein with a fidelity and efficiency rivaling the other 20 amino acids. The authors claim this is the first report of a completely self-sufficient bacterium capable of reading a 21 amino acid genetic code. It might also represent the first step in the creation of hybrid organisms capable of generating proteins with new or enhanced biological functions. Coauthor Christopher Anderson suggests that such organisms could produce ready-made “ketone- and polyethylene glycol–containing amino acids” suitable for pharmacological applications. AM
Authors at the National Institute of Dental and Craniofacial Research, National Institutes of Health (NIH; Bethesda, MD) have ingeniously engineered anthrax toxin so it specifically kills tumor cells and its general toxicity in mice is greatly attenuated (Proc. Natl. Acad. Sci. USA, 13 January 2003; 10.1073/pnas.0236849100). Anthrax toxin mediates its deadly action through three components: protective antigen, lethal factor, and edema factor. For toxin to act efficiently, protective antigen must be cleaved at the furin-activation sequence. The NIH researchers, led by Stephen Leppla and Thomas Bugge, set about investigating whether substitution of the furin-activation sequence in protective antigen with an artificial peptide sequence activated by urokinase would attenuate anthrax toxicity. Sure enough, the altered toxin was nontoxic to mice, even in very large doses (200 μg). As urokinase activity is a common hallmark of malignancy in cells, the researchers also tested anticancer activity of the engineered toxin in mice bearing established tumors. Animals receiving two toxin injections three days apart exhibited an 86–98% reduction in tumor size compared with controls. The authors suggest the hybrid anthrax toxin could have broad applicability for the treatment of human tumors, although repeated administration may elicit neutralizing antibody responses and it might be difficult to penetrate large solid tumor masses. AM
The preferred method for production of monoclonal antibodies is mammalian cell culture in large fermentation facilities; however, such systems are difficult to scale up and can be expensive. An ideal alternative system would be simple, scalable, containable, and economically attractive. To this end, researchers at the Scripps Research Institute (La Jolla, CA) have developed a novel platform to synthesize antibodies in algal chloroplasts, an approach with potential to meet all of the above demands (Proc. Natl. Acad. Sci. USA 100, 438–442, 2003). Richard Lerner and colleagues have expressed a large single-chain antibody against herpes simplex virus glycoprotein D in the chloroplast of the unicellular green alga Chlamydomonas reinhardtii. To optimize antibody production, they modified the antibody-coding sequence to reflect codon usage (that is, the genetic language specific to chloroplast proteins). Antibody expression was driven by chloroplast-specific promoters and ancillary control sequences. The ease with which green algae can be genetically engineered and grown, as well as their safety, make them an attractive alternative to current antibody production methodologies. GTO
Researchers at the Albert Einstein College of Medicine (Bronx, NY) have synthesized a derivative of ecdysone that can be activated by light and turn on gene expression in targeted tissues or cells (Chem. Biol. 9, 1347–1353, 2002). The insect molting hormone ecdysone is a steroid hormone that triggers gene expression by binding to and activating a nuclear dimer of the ecdysone receptor and a gene-specific receptor that in turn activates transcription of genes associated with an ecdysone responsive element. This control system is routinely used in gene expression of mammalian cells. Taking ecdysone as their starting material, David Lawrence and coworkers have now engineered a “caged” version of the hormone. This inactive ecdysone derivative can be readily photo-converted back to “free,” active ecdysone by a light beam. “Encaging” entailed a series of chemical transformation steps (acylation and alkylation under vigorous reaction conditions). Caged ecdysone is a highly permeable compound and thus diffuses easily into tissues and cells. Cell- and tissue-specific gene activation is achieved by targeting the desired sites with light beams. Spatially constrained gene activation mediated by spot illumination holds promise for discretely controlling protein expression in a multicellular setting. GTO
Research Briefs written by Andrew Marshall, Meeghan Sinclair, and Gaspar Taroncher-Oldenburg.